The present invention relates to methods and apparatus for mass spectrometry.
Tandem mass spectrometry (MS/MS) is the name given to the method of mass spectrometry wherein parent ions generated from a sample are selected by a first mass filter/analyser and are then passed to a collision cell wherein they are fragmented by collisions with neutral gas molecules to yield daughter (or xe2x80x9cproductxe2x80x9d) ions. The daughter ions are then mass analysed by a second mass filter/analyser, and the resulting daughter ion spectra can be used to determine the structure of the parent (or xe2x80x9cprecursorxe2x80x9d) ion. Tandem mass spectrometry is particularly useful for the analysis of complex mixtures such as biomolecules since it avoids the need for chemical clean-up prior to mass spectral analysis.
A particular form of tandem mass spectrometry referred to as parent ion scanning is known, wherein in a first step the second mass filter/analyser is arranged to act as a mass filter so that it will only transmit and detect daughter ions having a specific mass-to-charge ratio. The specific mass-to-charge ratio is set so as to correspond with the mass-to-charge ratio of daughter ions which are known to be characteristic products which result from the fragmentation of a particular parent ion or type of parent ion. The first mass filter/analyser upstream of the collision cell is then scanned whilst the second mass filter/analyser remains fixed to monitor for the presence of daughter ions having the specific mass-to-charge ratio. The parent ion mass-to-charge ratios which yield the characteristic daughter ions can then be determined. As a second step, a complete daughter ion spectrum for each of the parent ion mass-to-charge ratios which produce characteristic daughter ions may then be obtained by operating the first mass filter/analyser so that it selects parent ions having a particular mass-to-charge ratio, and scanning the second mass filter/analyser to record the resulting full daughter ion spectrum. This can then be repeated for the other parent ions of interest. Parent ion scanning is useful when it is not possible to identify parent ions in a direct mass spectrum due to the presence of chemical noise, which is frequently encountered, for example, in the electrospray mass spectra of biomolecules.
Triple quadrupole mass spectrometers having a first quadrupole mass filter/analyser, a quadrupole collision cell into which a collision gas is introduced, and a second quadrupole mass filter/analyser are well known. Another type of mass spectrometer (a hybrid quadrupole-time of flight mass spectrometer) is known wherein the second quadrupole mass filter/analyser is replaced by an orthogonal time of flight mass analyser.
As will be shown below, both types of mass spectrometers when used to perform conventional methods of parent ion scanning and subsequently obtaining a daughter ion spectrum of a candidate parent ion suffer from low duty cycles which render them unsuitable for use in applications which require a higher duty cycle i.e. when used in on-line chromatography applications.
Quadrupoles have a duty cycle of approximately 100% when being used as a mass filter, but their duty cycle drops to around 0.1% when then are used in a scanning mode as a mass analyser, for example, to mass analyse a mass range of 500 mass units with peaks one mass unit wide at their base.
Orthogonal acceleration time of flight analysers typically have a duty cycle within the range 1-20% depending upon the relative m/z values of the different ions in the spectrum. However, the duty cycle remains the same irrespective of whether the time of flight analyser is being used as a mass filter to transmit ions having a particular mass to charge ratio, or whether the time of flight analyser is being used to record a full mass spectrum. This is due to the nature of operation of time of flight analysers. When used to acquire and record a daugher ion spectrum the duty cycle of a time of flight analyser is typically around 5%.
To a first approximation the conventional duty cycle when seeking to discover candidate parent ions using a triple quadrupole mass spectrometer is approximately 0.1% (the first quadrupole mass filter/analyser is scanned with a duty cycle of 0.1% and the second quadrupole mass filter/analyser acts as a mass filter with a duty cycle of 100%). The duty cycle when then obtaining a daughter ion spectrum for a particular candidate parent ion is also approximately 0.1% (the first quadrupole mass filter/analyser acts as a mass filter with a duty cycle of 100%, and the second quadrupole mass filter/analyser is scanned with a duty cycle of approximately 0.1%). The resultant duty cycle therefore of discovering a number of candidate parent ions and producing a daughter spectrum of one of the candidate parent ions is approximately 0.1%/2 (due to a two stage process with each stage having a duty cycle of 0.1%)=0.05%.
The duty cycle of a quadrupole-time of flight mass spectrometer for discovering candidate parent ions is approximately 0.005% (the quadrupole is scanned with a duty cycle of approximately 0.1% and the time of flight analyser acts a mass filter with a duty cycle of approximately 5%). Once candidate parent ions have been discovered, a daughter ion spectrum of a candidate parent ion can be obtained with an duty cycle of 5% (the quadrupole acts as a mass filter with a duty cycle of approximately 100% and the time of flight analyser is scanned with a duty cycle of 5%). The resultant duty cycle therefore of discovering a number of candidate parent ions and producing a daughter spectrum of one of the candidate parent ions is approximately 0.005% (since 0.005% less than  less than 5%).
As can be seen, a triple quadrupole has approximately an order higher duty cycle than a quadrupole-time of flight mass spectrometer for performing conventional methods of parent ion scanning and obtaining confirmatory daughter ion spectra of discovered candidate parent ions. However, such duty cycles are not high enough to be used practically and efficiently for analysing real time data which is required when the source of ions is the eluent from a chromatography device.
Electrospray and laser desorption techniques have made it possible to generate molecular ions having very high molecular weights, and time of flight mass analysers are advantageous for the analysis of such large mass biomolecules by virtue of their high efficiency at recording a full mass spectrum. They also have a high resolution and mass accuracy.
Other forms of mass analysers such as quadrupole ion traps are similar in some ways to time of flight analysers, in that like time of flight analysers, they can not provide a continuous output and hence have a low efficiency if used as a mass filter to continuously transmit ions which is an important feature of the conventional methods of parent ion scanning. Both time of flight mass analysers and quadrupole ion traps may be termed xe2x80x9cdiscontinuous output mass analysersxe2x80x9d.
It is therefore desired to provide improved methods and apparatus for mass spectrometry, and according to a preferred embodiment to provide improved methods and apparatus which can identify candidate parent ions faster than conventional methods which would be suitable for use in chromatography applications on a real time basis.
According to a first embodiment and first aspect of the present invention, the first step of discovering candidate parent ions can be performed with a duty cycle of 2.5% (the quadrupole mass filter has a duty cycle of 100% and the time of flight analyser has a duty cycle of 5%, but two experimental runs need to be performed, one with the collision cell operated in a high fragmentation mode and the other with the collision cell operated in a low fragmentation mode, thereby halving the resultant duty cycle from 5% to 2.5%). The second step of confirming the identity of a particular candidate parent ion by performing a full daughter spectrum of the candidate parent ion can be performed with a duty cycle of 5% (the quadrupole again operates as a mass filter with approximately 100% duty cycle and the time of flight analyser acts as an analyser with a duty cycle of approximately 5%). Accordingly, only three experimental runs are required in order to discover a number of candidate parent ions and to produce a daughter ion spectrum of one of the candidate parent ions, each experimental run having a duty cycle of 5%. The resultant overall duty cycle is therefore 5%/3=1.67%.
The preferred embodiment therefore has a duty cycle which is approximately 30 times better than that of the conventional method performed on a triple quadrupole arrangement, and shows an improvement greater than 300 times compared with the conventional method performed on a quadrupole-time of flight mass spectrometer. Such an improvement enables the apparatus and method according to the preferred embodiment to used effectively at on-line chromatography time scales.
When the fragmentation means is operated in the first mode, a high voltage is applied to the fragmentation means which causes the ions passing therethrough to fragment. However, when the fragmentation means is operated in the second mode then the ions are substantially less fragmented and there is a higher proportion of molecular ions which are transmitted therethrough.
Preferably, operating the fragmentation means in the first mode comprises the step of supplying a voltage to the fragmentation means selected from the group comprising: (i) xe2x89xa715V; (ii) xe2x89xa720V; (iii) xe2x89xa725V; (iv) xe2x89xa730V; (v) xe2x89xa750V; (vi) xe2x89xa7100V; (vii) xe2x89xa7150V; and (viii) xe2x89xa7200V. Preferably, operating the fragmentation means in the second mode comprises the step of supplying a voltage to the fragmentation means selected from the group comprising: (i) xe2x89xa65V; (ii) xe2x89xa64.5V; (iii) xe2x89xa64V; (iv) xe2x89xa63.5V; (v) xe2x89xa63V; (vi) xe2x89xa62.5V; (vii) xe2x89xa62V; (viii) xe2x89xa61.5V; (ix) xe2x89xa61V; (x) xe2x89xa60.5V; and (xi) substantially OV. However, according to less preferred arrangements for both the first and second embodiments of the present invention, a voltage between 5V and 15V could be used for the first mode and/or the second mode. In such circumstances it would be expected that a proportion of the ions in the high energy mode would not actually be fragmented and similarly, in the low energy mode, a proportion of the ions would be fragmented.
In order to filter the ions, a first mass filter upstream of a fragmentation means, e.g. a collision cell, is preferably arranged so that only ions having a mass-to-charge ratio (hereinafter xe2x80x9cm/zxe2x80x9d) greater than a certain m/z are transmitted i.e. according to a preferred embodiment the first mass filter is initially set to operate as a high pass filter. The cutoff point may be set so that it is a little higher than the m/z value of the characteristic daughter ion which is being monitored for. For example, if the characteristic daughter ion is known to have a m/z value of 300, then the first mass filter may be set to only transmit ions having a m/z greater than say 350. Therefore, if an ion having a m/z value of 300 is subsequently detected by the mass analyser, then it follows that the ion must be a daughter ion caused by fragmentation of a parent ion in the fragmentation means since parent ions having this m/z would be filtered out by the first mass filter.
Preferably, the first range is variable. The range of ions transmitted by the first mass filter can therefore be altered every scan if necessary.
Preferably, the step of mass analysing at least some of the ions which have passed through the fragmentation means operating in the first mode comprises obtaining a first mass spectrum and wherein the step of mass analysing at least some of the ions which have passed through the fragmentation means operating in the second mode comprises obtaining a second mass spectrum.
Preferably, after the step of mass analysing at least some of the ions which have been passed through the fragmentation means operating in the second mode, the method further comprises the step of identifying at least one candidate parent ion. The at least one candidate parent ion is preferably identified by comparing the intensity of ions having a certain mass-to-charge ratio in the first mass spectrum with the intensity of ions having the same mass-to-charge ratio in the second mass spectrum. If a high intensity peak is found in the low energy spectrum but not in the high energy spectrum then it is likely that the peak represents a candidate parent ion.
Preferably, the method further comprises the steps of: filtering the ions upstream of the fragmentation means so that ions having a mass-to-charge ratio within a second range which includes at least one candidate parent ion are arranged to be substantially transmitted to the fragmentation means and so that the transmission of ions having a mass-to-charge ratio outside of the second range is substantially reduced; operating the fragmentation means so that substantially more of the ions are fragmented than in the second mode; and then mass analysing at least some of the ions which have passed through the fragmentation means. In otherwords, once a candidate parent ion has been identified, then the first mass filter is preferably set to operate as a narrow bandpass filter substantially only allowing ions at the m/z value of a particular candidate parent ion to be transmitted. According to a preferred embodiment, the second range is selected so that only ions having mass-to-charge ratios within xc2x1x mass-to-charge units of a candidate parent ion are substantially transmitted to the fragmentation means (4), wherein x is selected from the group comprising: (i) 0.5; (ii) 1.0; (iii) 2.0; (iv) 5.0; (v) 10.0; (vi) 15.0; and (vii) 20.0. The mass spectrometer therefore operates in a tandem MS mode.
Preferably, the ion source is selected from the group comprising: (i) an electrospray ion source; (ii) an atmospheric pressure chemical ionization ion source; and (iii) a matrix assisted laser desorption ion source. Such ion sources, especially the first two, may be provided with an eluent over a period of time, the eluent having been separated from a mixture by means of liquid chromatography.
Preferably, the ion source is selected from the group comprising: (i) an electron impact ion source; (ii) a chemical ionization ion source; and (iii) a field ionisation ion source. Such ion sources may be provided with an eluent over a period of time, the eluent having been separated from a mixture by means of gas chromatography.
Preferably, the mass analysing steps are performed by an analyser selected from the group comprising: (i) a quadrupole mass filter; (ii) a time-of-flight mass analyser; (iii) an ion trap; (iv) a magnetic sector analyser; and (v) a Fourier Transform Ion Cyclotron Resonance (xe2x80x9cFTICRxe2x80x9d) mass analyser. A time-of-flight mass analyser is particularly preferred.
Preferably, the filtering step(s) are performed by a multi-element ion optical lens, preferably a quadrupole rod set, which is further preferably provided with both a RF and a DC electric field.
Preferably, the multi-element ion optical lens is arranged to substantially transmit only ions having mass-to-charge ratios greater than a first value. Further preferably, the first value is selected from the group comprising: (i) 100; (ii) 150; (iii) 200; (iv) 250; (v) 300; (vi) 350; (vii) 400; (viii) 450; and (ix) 500. The step of identifying daughter ions in a preferred embodiment comprises identifying at least some ions which are determined to have mass-to-charge ratios less than the first value.
Preferably, the fragmentation means comprises a collision cell selected from the group comprising: (i) a quadrupole rod set; (ii) an hexapole rod set; (iii) an octopole rod set; and (iv) an electrode ring set. Further preferably, the collision cell is operated in a RF only mode and in a preferred arrangement is provided with a collision gas at a pressure within the range 10xe2x88x924 to 10xe2x88x921 mbar, preferably 10xe2x88x923 to 10xe2x88x922 mbar. Further preferably, the collision cell forms a substantially gas-tight enclosure. The collision gas may preferably comprise helium, argon, nitrogen, air or methane.
Preferably, the predetermined daughter ions comprises ions selected from the group comprising: (i) immonium ions from peptides; (ii) functional groups which includes, for example, phosphate group PO3xe2x88x92 ions from phosphorylated peptides; and (iii) mass tags which are intended to cleave from a specific molecule or class of molecule and to be subsequently identified thus reporting the presence of the specific molecule or class of molecule.
According to a preferred embodiment it is possible to search for candidate parent ions by interrogating the high collision energy MS spectrum (i.e. daughter ion spectrum) for more than one characteristic daughter ion. This may be particularly relevant when the parent ions have been xe2x80x9ctaggedxe2x80x9d with a specific mass tag. A mixture of two or more parent ions may be tagged each with a different mass tag and which could be discovered by simultaneously monitoring for two or more characteristic daughter ions. Hence, parent ions from two or more different classes of compounds could be discovered in the same set of experiments.
The implementation of the various steps by a control system, preferably on automatic control system, is merely a preferred feature. In a less preferred embodiment some of the method steps could involve human interaction from an operator.
Whereas in the first embodiment, the fragmentation means was operated in the second mode (where there was a lesser degree of fragmentation) only once a daughter ion of interest had been identified, according to the second embodiment the fragmentation means preferably switches back and forth between the high and low energy modes i.e. a parent ion spectrum may be obtained without having first determined (or irrespective of) whether, for example, a predetermined daughter ion has been determined to be present.
Three different modes of operation (or sub-embodiments) are contemplated by the second embodiment. In a first mode of operation it is only necessary to determine whether a predetermined daughter ion is present in the daughter ion spectrum. In this particular mode it is not strictly necessary for a candidate parent ion to have first been identified, although this is preferable. In a second mode of operation it is determined whether there could be some connection between at least one daughter ion and at least one candidate parent ion by virtue of the loss of a predetermined ion (such as, for example, a functional group) or the loss of a neutral particle. A third mode of operation is also contemplated in which the determining steps of both the first and second modes of operation may be performed.
Preferably, the method further comprises the step of filtering the ions upstream of the fragmentation means so that ions having a mass-to-charge ratio within a first range are substantially transmitted and so that the transmission of ions having a mass-to-charge ratio outside of the first range is substantially reduced.
Preferably, the first range is variable and hence may be altered each scan.
Preferably, the step of identifying at least one daughter ion comprises determining at least some ions which have a mass-to-charge ratio which falls outside of the first range. According to the second embodiment, identifying a daughter ion on the basis of the daughter ion having a m/z lower than the cut-off value of a first mass filter is only one way of identifying a daughter ion. Other ways of identifying a daughter ion are also contemplated.
Preferably, the step of mass analysing at least some of the ions which have passed through the fragmentation means operating in the first mode comprises obtaining a first mass spectrum and wherein the step of mass analysing at least some of the ions which have passed through the fragmentation means operating in the second mode comprises obtaining a second mass spectrum.
Preferably, the at least one candidate parent ion is identified by comparing the intensity of ions having a certain mass-to-charge ratio in the first (daughter ion) mass spectrum with the intensity of ions having the same mass-to-charge ratio in the second (parent ion) mass spectrum. Preferably, the at least one daughter ion is identified by comparing the intensity of ions having a certain mass-to-charge ratio in the first mass spectrum with the intensity of ions having the same mass-to-charge ratio in the second mass spectrum. A candidate parent ion will preferably have a much higher intensity in said second mass spectrum compared with said first spectrum (and vice versa for a daughter ion).
Preferably, if it is determined that: (i) the at least one daughter ion corresponds with a predetermined daughter ion; and/or (ii) the at least one daughter ion and the at least one candidate parent ion could be related by the loss of a predetermined ion or neutral particle, then the method further comprises the steps of: filtering the ions upstream of fragmentation means so that ions having a mass-to-charge ratio within a second range which includes at least one candidate parent ion are arranged to be substantially transmitted to the fragmentation means and so that the transmission of ions having a mass-to-charge ratio outside of the second range is substantially reduced; operating the fragmentation means so that substantially more of the ions are fragmented than in the second mode; and mass analysing at least some of the ions which have passed through the fragmentation means. In other words once a daughter ion of interest or an interesting connection or relationship between a parent ion and a daughter ion has been established, then the mass spectrometer switches to operate in a tandem MS mode.
Preferably, the second range is selected so that only ions having mass-to-charge ratios within xc2x1x mass-to-charge units of a candidate parent ion are substantially transmitted to the fragmentation means, wherein x is selected from the group comprising: (i) 0.5; (ii) 1.0; (iii) 2.0; (iv) 5.0; (v) 10.0; (vi) 15.0; and (vii) 20.0. The mass filter upstream of the collision cell therefore preferably operates as a narrow bandpass filter.
Preferably, the ion source is selected from the group comprising: (i) an electrospray ion source; (ii) an atmospheric pressure chemical ionization ion source; and (iii) a matrix assisted laser desorption ion source. Preferably, such an ion source, especially the first two, is provided with an eluent over a period of time, the eluent having been separated from a mixture by means of liquid chromatography.
Preferably, the ion source is selected from the group comprising: (i) an electron impact ion source; (ii) a chemical ionization ion source; and (iii) a field ionisation ion source. Preferably, such an ion source is provided with an eluent over a period of time, the eluent having been separated from a mixture by means of gas chromatography.
Preferably, the mass analysing steps are performed by an analyser selected from the group comprising: (i) a quadrupole rod set; (ii) a time-of-flight mass analyser; (iii) an ion trap; (iv) a magnetic sector analyser; and (v) a Fourier Transform Ion Cyclotron Resonance (xe2x80x9cFTICRxe2x80x9d) mass analyser. A time-of-flight mass analyser is particularly preferred.
Preferably, the filtering step(s) are performed by a multi-element ion optical lens, preferably a quadrupole mass filter. Further preferably, both a RF and a DC electric field are applied to the multi-element ion optical lens.
Preferably, the multi-element ion optical lens is arranged to substantially transmit only ions having mass-to-charge ratios greater than a first value. Further preferably, the first value is selected from the group comprising: (i) 100; (ii) 150; (iii) 200; (iv) 250; (v) 300; (vi) 350; (vii) 400; (viii) 450; and (ix) 500. Preferably, the step of identifying daughter ions comprises identifying at least some ions which are determined to have mass-to-charge ratios less than the first value.
Preferably, the fragmentation means comprises a collision cell selected from the group comprising: (i) a quadrupole rod set; (ii) an hexapole rod set; (iii) an octopole rod set; and (iv) an electrode ring set. Preferably, the collision cell is operated in a RF only mode, and is further preferably provided with a collision gas at a pressure within the range 10xe2x88x923 to 10xe2x88x921 mbar, preferably 10xe2x88x923 to 10xe2x88x922 mbar. Preferably, the collision cell forms a substantially gas-tight enclosure.
Preferably, the predetermined daughter ions comprises ions selected from the group comprising: (i) immonium ions from peptides; (ii) functional groups including phosphate group PO3xe2x88x92 ions from phosphorylated peptides; and (iii) mass tags which are intended to cleave from a specific molecule or class of molecule and to be subsequently identified thus reporting the presence of the specific molecule or class of molecule.
Preferably, operating the fragmentation means in the first mode comprises the step of supplying a voltage to the fragmentation means selected from the group comprising: (i) xe2x89xa715V; (ii) xe2x89xa720V; (iii) xe2x89xa725V; (iv) xe2x89xa730V; (v) xe2x89xa750V; (vi) xe2x89xa7100V; (vii) xe2x89xa7150V; and (viii) xe2x89xa7200V.
Preferably, operating the fragmentation means in the second mode comprises the step of supplying a voltage to the fragmentation means selected from the group comprising: (i) xe2x89xa65V; (ii) xe2x89xa64.5V; (iii) xe2x89xa64V; (iv) xe2x89xa63.5V; (v) xe2x89xa63V; (vi) xe2x89xa62.5V; (vii) xe2x89xa62V; (viii) xe2x89xa61.5V; (ix) xe2x89xa61V; (x) xe2x89xa60.5V; and (xi) substantially OV.
Although it is preferred in the first embodiment (and optionally in the second embodiment) for the quadrupole mass filter to have initially a high pass characteristic, in less preferred embodiments the mass filter may have a bandpass characteristic. It is also contemplated in less preferred embodiments that the mass filter could have a xe2x80x9cV-notchedxe2x80x9d transmission profile i.e. high transmission at low and high mass-to-charge ratios and preferably linearly or otherwise rapidly decreasing/increasing transmission either side of a mid-point.
The implementation of alternating low and high collision energy in both the first and second embodiments allows for (candidate) parent ions to be selected based on the occurrence of a specific daughter ion m/z value, either nominal or exact, in the high collision energy xe2x80x9cMS surveyxe2x80x9d spectrum. According to the second embodiment, the selection criteria may also include selection based on the occurrence of ions with a specific difference in m/z value, either nominal or exact, between those in the low and high collision energy xe2x80x9cMS surveyxe2x80x9d spectra.
Once one or more parent ions have been discovered, then according to both embodiments, a number of further criteria may be used for the further selection and/or rejection of candidate parent ions i.e. to refine the list of possible candidate parent ions down to a shortlist of more definite candidate parent ions. These criteria include:
(a) selection based on required charge state (typically Z greater than 1 for peptides, Z=1 for drug metabolites);
(b) selection based on relative or absolute intensity;
(c) selection based on inclusion within a preferred m/z range;
(d) selection based on list of preferred m/z values, either nominal or exact;
(e) rejection based on list of excluded m/z values, either nominal or exact (typically known background ions or matrix related impurities);
(f) rejection based on temporary (dynamic) list of excluded m/z values (typically precursor ions that have recently been analysed to prevent duplication).
According to the second embodiment and as a less preferred feature of the first embodiment, daughter ions formed by the fragmentation of multiply charged parent ions may be detected by the presence of ions having mass-to-charge ratios higher than the mass-to-charge ratios of candidate parent ions. This may be particularly appropriate when parent ions are generated by electrospray.
According to the first and second embodiment, in the event of multiple co-eluting components the true precursor ion may be discovered by using the first mass filter, MS1, to select each candidate precursor ion in turn to record its MS/MS fragment spectrum. However, the number of spectra to be acquired will only be increased by a number equal to just the number of candidate precursor ions. This is still much less than the many hundreds of spectra required by traditional parent ion scanning methods.
In the case of multiple co-eluting components there is scope for reducing the number of candidate precursor ions by the use of additional filtering criteria. For example, the targeted precursor ion may be discovered if the high collision energy spectrum is also interrogated for the presence of one or more characteristic neutral loss ions corresponding to each of the candidate precursor ions observed in the low collision energy spectrum. This may reduce the number of MS/MS fragment spectra to be recorded, in many cases to just one spectrum.
In principal, if the number of candidate precursor ions is four or more the number of MS/MS spectra to be acquired could be further reduced by repeatedly sub-dividing the candidate precursors in two equal or near equal sub-groups according to their mass. The high collision energy spectrum for all the precursor ions within each sub-group would then be recorded by setting the low-mass cut-off for MS1 to a m/z value dividing the two groups. By a process of elimination this procedure would allow arrival at the targeted precursor ion in less stages. In practice, this approach is preferred only when the number of candidate precursor ions is six or more. Nevertheless, to illustrate the potential value of this method, a mixture of 16 components may require 16 MS/MS spectra to discover the target precursor ion, whereas this approach could reduce the required number of MS/MS spectra to five.
Precursor ion discovery based on the presence of a specific product ion m/z value requires initial interrogation of only the high energy CID (Collision Induced Decomposition) xe2x80x9cMS surveyxe2x80x9d spectra. If appropriate, the m/z transmission range of the quadrupole mass filter may be set such as not to transmit the m/z value of the specified product ion, thereby removing any background ions from the source at that m/z value. Any ions at the specified m/z value can only be product ions. When a daughter ion of interest elutes, the low energy CID xe2x80x9cMS surveyxe2x80x9d spectrum now yields a short list of (candidate) parent ions. This list may optionally be further filtered or refined by various selection and/or rejection criteria, such as charge state, excluded m/z values, etc. Confirmation and identification of the targeted precursor ion now only requires acquisition of MSxe2x80x94MS spectra for the (optionally further filtered) short list of candidates. This achieves the same goal as traditional parent ion scanning without the need to scan the first mass filter, MS1, and with the added bonus of having acquired the full daugher ion spectrum of the targeted precursor ion. Specification of exact product ion m/z values further enhances selectivity.
Precursor Ion Discovery based on the presence of a specific neutral or ion loss requires interrogation of both the low and high energy CID xe2x80x9cMS surveyxe2x80x9d spectra. The low energy spectra yield a short list of candidate precursor ions. Again this short list may be further filtered by various criteria, i.e. charge state, excluded m/z values, etc. A short list of m/z values with the specified neutral or ion loss may now be generated. These m/z values are now searched against the high energy CID xe2x80x9cMS surveyxe2x80x9d spectrum. The precursor ion for any hits may be confirmed and identified by acquisition of its MSxe2x80x94MS spectrum. This achieves the same goal as traditional neutral loss scanning without the need to scan MS1 and MS2, and again with the added bonus of having acquired the full product ion spectrum of the targeted precursor ion. Again exact m/z values may be specified.
The various preferred embodiments provide numerous advantages over conventional techniques of parent ion scanning, including the possibility of discovering the mass-to-charge ratios of parent ions and to obtain their corresponding daughter ion spectra within on-line time scales e.g. chromatography time scales. The preferred embodiments also have higher sensitivities than conventional parent ion scanning methods, and open up the possibility of incorporating multiple criteria into the same experiment for selection of parent ion m/z values. It is also possible to discover multiple classes of parent ion within the same experiment and the methods can be used with mass tagging.